Polyanionic polymer adjuvants for haemophilus influenzae b saccharide vaccines

ABSTRACT

The present invention relates to immunogenic compositions comprising capsular polysaccharide or oligosaccharide of  H. influenzae  B (PRP) and methods of making such compositions.

This application is a continuation of U.S. application Ser. No.13/396,843, filed Feb. 15, 2012, which is a continuation of U.S.application Ser. No. 12/785,809, filed May 24, 2010, now U.S. Pat. No.8,137,681, issued Mar. 20, 2012, which is a continuation of U.S.application Ser. No. 10/560,513, filed Dec. 13, 2005, now U.S. Pat. No.8,007,818, issued Aug. 30, 2011, which was the U.S. National StageApplication of International Application No. PCT/EP2004/006426, filedJun. 14, 2004, which claims priority to United Kingdom Application No.GB 0313916.9, filed Jun. 16, 2003, the entire contents of which areincorporated herein.

The present invention relates to the field of vaccines, and, inparticular, to immunogenic compositions comprising antigens of lowisoelectric point such as H. influenzae B capsular polysaccharide oroligosaccharide (PRP). The present invention presents immunogeniccompositions and combination vaccines comprising PRP wherein the PRP isprotected to some degree from immune interference that can occur whenPRP is combined with other antigen formulations—particularlyformulations comprising DTPa; a well known ‘trivalent’ combinationvaccine comprising Diphtheria toxoid (DT), tetanus toxoid (TT), andacellular B. pertussis components [typically comprising detoxifiedpertussis toxoid (PT) and filamentous haemagglutinin (FHA) with optionalpertactin (PRN) and/or agglutinogens 2 and 3], typically adsorbed (atleast in part) on aluminium hydroxide adjuvant, for example the marketedvaccine INFANRIX-DTPa™ (GlaxoSmithKline Biologicals) which contains DT,TT, PT, FHA, and PRN antigens, all adsorbed onto aluminium hydroxideadjuvant. A method to reduce interference of PRP in a combinationvaccine (comprising, for instance, DTPa) is also presented.

Vaccines that utilise polysaccharides are known in the art. For examplea PRP vaccine for the prevention of Haemophilus influenzae B infectionsis based on the H. influenzae B capsular oligosaccharide orpolysaccharide (PRP) conjugated with a carrier protein. Thepolysaccharide is a polymer of ribose, ribitol and phosphate. Examplesof carrier protein include diphtheria or tetanus toxoid, or an outermembrane protein of N. meningitidis. See for example U.S. Pat. No.4,365,170, U.S. Pat. No. 4,673,574, EP 208375, EP 477508 and EP 161188.

It is desirable to administer such conjugate vaccines with otherantigens or vaccines at the same time and this can involve multipleinjections. Problems associated with multiple injections include a morecomplicated administration procedure and a large total injection volume.This is a particularly acute problem when the vaccine is intended forinfants. For both the infant and the practitioner it is desirable toinject all necessary antigens in one shot of normal volume, thusrendering the vaccination procedure less traumatic and painful for theinfant, and more efficient and easier to manage for the practitioner.

It has therefore been proposed to combine such polysaccharide conjugatevaccines with other vaccines such as DTPa or DTPw (where the pertussiscomponent is killed whole-cell Bordetella pertussis) to produce moreelaborate combination vaccines. In addition, the inclusion of furtherantigens to such a combination vaccine for the prevention of diseaseslike hepatitis B or Polio has also been proposed (combination vaccinescomprising an antigen against hepatitis B and antigens againstdiphtheria, tetanus and pertussis (HepB, DTPa) have been described in WO93/24148). See also WO 98/00167 and WO 99/13906 which also discloseDTP-PRP combination vaccines.

It has been found, however, that simple mixing of the components of acombination vaccine is complicated by the fact that not all antigens canbe effectively mixed together. The reduction in the immunogenicity of anantigen when combined with other components (as compared to theparticular antigen administered alone) is known as interference. It isknown, for example, that the extemporaneous mixing of a DTPa combinationvaccine with unadjuvanted PRP conjugates results in a reduction ofantibody titres to the PRP polysaccharide (WO 97/00697). In addition, WO97/00697 showed that if PRP conjugate is adsorbed onto aluminiumhydroxide, there is a significant reduction of antibody titres to thepolysaccharide component. These results indicated that there wasinterference between the aluminium hydroxide of the DTPa vaccine andPRP. In order to try and minimise this interference in such anextemporaneously-prepared combination vaccine PRP was pre-adsorbed ontoaluminium phosphate.

Without wishing to be bound by theory, it is thought that the aboveinterference problem may be as a result of PRP (with a low isoelectricpoint of less than 2) forming a strong interaction with aluminiumhydroxide (with a high isoelectric point). This interaction may mask PRPepitopes from immune competent cells—particularly if the PRP/AlOHinteraction forms a network of particles—a phenomenon calledflocculation (see FIG. 5) which may be observed visually or by opticalmicroscope.

WO 96/37222 also describes the interference problem. In this case theantigenicity of PRP conjugate is stabilised by adsorbing it and theother DTPa components onto an aluminium-based adjuvant with a zero pointcharge (ZPC) of less than 7.2, for instance aluminium phosphate, oraluminium hydroxide to which anion salts have been added to lower itszero point charge from around 10 or 11 to under 7.2.

A problem with using aluminium phosphate entirely for a combinationvaccine is that many antigens in a combination vaccine benefitimmunologically from being adsorbed onto aluminium hydroxide—forinstance pertactin. Many of these antigens (for instance pertactin)cannot be adequately adsorbed onto aluminium phosphate, and becomedesorbed from aluminium hydroxide if sufficient anion salts are added toreduce its zero point charge under 7.2. Pertactin is one of the mostimportant components in the pertussis vaccine. Without wishing to bebound by theory, a significant lowering of its adsorption on adjuvantcould lead to a reduction of the T-cell response and the potency of theacellular pertussis vaccine as a whole. At pH 6.1 (the typical pH ofDTPa vaccines) a 24 hour adsorption step allows more than 90% ofpertactin to be adsorbed onto aluminium hydroxide, but less than 50% tobe adsorbed onto aluminium phosphate (reducing further when it iscombined with other antigens).

There is therefore a technical problem in combination vaccinescomprising PRP and antigens adsorbed onto aluminium hydroxide to reduceinterference to PRP, yet maintain a significant degree of adsorption ofantigens beneficially associated with aluminium hydroxide. A furthertechnical problem is that antigens of low pI can form aggregates (orflocculate) with adjuvant particles making the vaccine unsuitable foruse.

WO 99/48525 provides one solution to this problem which involves acomplex process of adsorbing and mixing antigens in order for PRP to beadded with minimised interference.

There is still need, however, for further solutions to the aboveproblems which are advantageously simpler—i.e. involve a single, simpleadditional process step, or involve the simple addition of a singlePRP-protective excipient to the immunogenic composition. The presentinvention provides such a solution.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Pre-saturation of Al(OH)₃ with PLG (106 residues).

FIG. 2: Competition between PRP-T and PLG (85 residues).

FIG. 3: Evaluation of the Hib-PLG formulations in the rabbit model.

FIG. 4: Evaluation of immunogenicity and impact of PLG on new PLG Hibformulations administered with INFANRIX PENTA™ in the infant rat model.

FIG. 5: Schematic representation of adsorption and flocculation.

FIG. 6: Scheme of clinical trial.

FIG. 7: Optical microscope picture of INFANRIX PENTA™ (DTPaHepBIPV).

FIG. 8: Optical microscope picture of sample flocculating when 5 μgsaccharide of PRP-TT is added to the sample.

FIG. 9: Optical microscope picture showing no flocculation in thepresence of 200 μM PLG Mw 2,200 (Sigma).

FIG. 10: Optical microscope picture showing no observable flocculationsin DTPw controls.

FIG. 11: Optical microscope picture showing observable flocculations inDTPw samples with reconstituted MenACHib-TT.

FIG. 12: Optical microscope picture showing that DTPwHepB/MenACHib with250 μM PLG exhibits reduced flocculation.

DESCRIPTION OF THE INVENTION

This invention relates to a general method by which eitherextemporaneously-prepared or liquid PRP/DTPa combination vaccines (orPRP/DTPw) can be made in order to reduce the PRP interference problemwhilst being able to maintain a significant degree of adsorption ofantigens beneficially associated with the aluminium-based adjuvant onwhich it is most immunogenic. In so doing, pertussis antigens incombination vaccines of the present invention may be stably retained intheir most potent form. The invention further provides immunogeniccompositions, vaccines and combination vaccines comprising PRP which isprotected to some degree from immune interference. The inventors havefound that the above can surprisingly be achieved by incorporating apolyanionic polymer excipient with the vaccine comprising PRP. Withoutwishing to be bound by theory, the polyanionic polymer can compete withPRP, protecting it from any aluminium hydroxide present in the vaccine(for example by reducing the amount or rate of binding of PRP toadjuvant and/or the extent or rate of flocculation), yet surprisinglydoes not cause antigens already adsorbed to aluminium hydroxide tobecome significantly desorbed.

Accordingly in one embodiment the present invention provides animmunogenic composition comprising a capsular polysaccharide oroligosaccharide of Haemophilus influenzae B (PRP), and a polyanionicpolymer.

Although PRP is described throughout this specification, it is envisagedthat the same solution may protect other oligosaccharides orpolysaccharides (or antigens in general) with a low isoelectric point(less than 4 or less than 3, preferably less than 2), and thereforewherever PRP is mentioned herein, such other oligosaccharides,polysaccharides or antigens may alternatively be included as part of theinvention. The isoelectric point (pI) above is that of the saccharidemoiety before any conjugation event. In particular capsularpolysaccharide or oligosaccharide isolated from N. meningitidisserogroup A or C may alternatively be used, the polyanionic polymer alsofunctioning to prevent, for instance, potential aggregation and/orimmunogenicity reduction events in the vaccines (for instance based onDTPa or DTPw combinations) of the invention (see Example 2). Thus animmunogenic composition comprising an antigen (preferably saccharide)with a low pI and a polyanionic polymer is envisioned.

By “oligosaccharide” and “polysaccharide” it is meant a saccharideepitope that has been isolated from a pathogen (for instance from abacterial cell's capsular polysaccharide). It may be isolated directlyfrom the bacterium, or may be processed in some way before use in thevaccines of the invention; for instance it may have been reduced in sizeby known techniques such as microfluidisation (see EP 497524 for othersuch size reduction techniques). The term “polysaccharide oroligosaccharide” throughout this specification may therefore be replacedby the term “saccharide”.

By “protection” of an antigen with low pI from adjuvants with high pI(such as AlOH) it is meant any or all of the following: a) that theantigen exhibits reduced binding to the adjuvant if the polyanionicpolymer is present, and/or b) that flocculation between antigen andadjuvant is reduced (preferably prevented) if the polyanionic polymer ispresent (for instance assessed by using optical microscopy techniques inExample 2, FIGS. 8 and 11 showing flocculated samples, and FIGS. 7, 9,10, 12 showing non-flocculated samples), and/or c) immune interferenceto the antigen is reduced (and preferably prevented) if the polyanionicpolymer is present (as measured by ELISA tests to establish GMT or GMCof anti-antigen antibody in sera of immunised animals).

The terms “comprising”, “comprise” and “comprises” herein is intended bythe inventors to be substitutable with the terms “consisting of”,“consist of” and “consists of”, respectively, in every instance.

In a preferred embodiment, PRP (or antigen of low pI) is conjugated to acarrier protein which is a source of T-helper cell epitopes.

Preferred carrier proteins for the polysaccharide or oligosaccharideconjugates of the present invention are tetanus toxoid, diphtheriatoxoid, CRM197, an outer membrane protein from a bacteria such as N.meningitidis, and protein D from non-typeable H. influenzae (EP594610).Most preferably, PRP (or other saccharide antigens of low pI) isconjugated to tetanus toxoid. The synthesis of Haemophilus influenzaetype B capsular polysaccharide (PRP) tetanus toxoid (TT) conjugate isdescribed, for example, in WO 97/00697.

The polysaccharide or oligosaccharide conjugates of the invention may beprepared by any known coupling technique. For example the polysaccharidecan be coupled via a thioether linkage. This conjugation method relieson activation of the polysaccharide with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) to form a cyanate ester. Theactivated polysaccharide may thus be coupled directly or via a spacergroup to an amino group on the carrier protein. Preferably, the cyanateester is coupled with hexane diamine and the amino-derivatisedpolysaccharide is conjugated to the carrier protein using heteroligationchemistry involving the formation of the thioether linkage. Suchconjugates are described in PCT published application WO93/15760(Uniformed Services University).

The conjugates can also be prepared by direct reductive aminationmethods as described in U.S. Pat. No. 4,365,170 (Jennings) and U.S. Pat.No. 4,673,574 (Anderson). Other methods are described in EP 161188, EP208375 and EP 477508.

A further method involves the coupling of a cyanogen bromide activatedpolysaccharide derivatised with adipic acid hydrazide (ADH) to theprotein carrier by carbodiimide condensation. Such conjugation isdescribed in Chu C. et al. Infec. Immunity, 1983 245 256.

A polyanionic polymer of the present invention is a polymer which, whendissolved in an aqueous medium at pH7 (and preferably also at pH 6.1—atypical pH of the DTPa and DTPw vaccines of the invention), isnegatively-charged due to the presence of anionic constitutionalrepeating units (for example, units containing sulphate, sulphonate,carboxylate, phosphate and borate groups). A constitutional repeatingunit or monomer refers to the minimal structural unit of a polymer. Thepolyanionic polymer may be a polyanionic heteropolymer, comprising twoor more different anionic constitutional repeating units, or may be apolyanionic homopolymer, consisting of a single anionic constitutionalrepeating unit. Although preferred, clearly not every monomer/repeatunit need be negatively charged as long as there is sufficient negativecharge for the polyanionic polymer of the invention to have thecapabilities stated herein, in particular the ability to preventflocculation between PRP and aluminium hydroxide adjuvant and/or thecapability not to significantly desorb antigens beneficially adsorbed toaluminium hydroxide.

The polyanionic polymer of the invention may be a chemical polymer andmay comprise anionic constitutional repeating units obtained from agroup consisting of: acrylic acid, methacrylic acid, maleic acid,fumaric acid, ethylsulphonic acid, vinylsulphonic acid, vinylsulphonicacid, styrenesulphonic acid, vinylphenylsulphuric acid,2-methacryloyloxyethane sulphonic acid,3-methacryloyloxy-2-hydroxypropanesulphonic acid, 3-methacrylamido-3-methylbutanoic acid, acrylamidomethylpropanesulfonic acid,vinylphosphoric acid, 4-vinylbenzoic acid, 3-vinyloxypropane-1-sulphonic acid, N-vinylsuccinimidic acid, and salts of theforegoing.

Alternatively, the polyanionic polymer of the invention may (or may not,as this is not a preferred embodiment) be an oligo- or poly-saccharidesuch as dextran.

Most preferably, the polyanionic polymer of the invention is an oligo-or poly-peptide. Such peptides may be D- or L-peptides (preferably thelatter so that peptide is advantageously bio-degradable), and maycomprise anionic constitutional repeating units (or monomers) such asL-aspartic acid, D-aspartic acid, L-glutamic acid, D-glutamic acid,non-natural anionic amino acids (or salts or anionic chemicalderivatives thereof).

For the purposes of this invention, the polyanionic polymer of thepresent invention is an oligo- or poly-peptide which has a monomercontent of no less than 30, 40, 50, 60, 70, 80, 90 or, preferably, 100%L-aspartic acid and/or L-glutamic acid.

Preferably the polyanionic polymer of the invention consists of, onaverage, 4-200 or 5-200 monomers, preferably 8-117 monomers, morepreferably 15-32 or 15-18 monomers, most preferably 17 monomers (orresidues in the case of peptides). As polymers are complex populationsof molecules of potentially different lengths, “on average” means thatnumber of monomers or residues is calculated according to theweight-average molecular weight (or M_(w)) of the polyanionic polymermeasured by MALLS divided by the molecular weight of the monomer.Preferably the polydispersity (a measure of homogeneity) of thepolyanionic polymer is less than 3. Multiple angle laser lightscattering (MALLS) is a well known technique for obtaining M_(w) andpolydispersity measurements of polymers (and is typically carried outusing a TSKG3000PWXL HPLC column at flow rate 0.75 ml/min, eluted in 10mM phosphate buffer pH 7.6, 130 mM NaCl). It is also envisaged that thepolyanionic polymer of the invention may comprise mixtures of 2 or morepolyanionic polymers with different M_(w)s. For instance blends of 16mers and 8 mers or 16 mers and 4 mers of the same type of polymer may beadvantageously used for the purposes of this invention.

Without wishing to be bound by theory, it is thought that polyanionicpolymers have advantages over using anions or anionic monomers, asanions have to be present at a high concentration in a vaccine toprevent PRP binding and/or flocculating with AlOH adjuvant. At such highconcentrations antigens beneficially adsorbed onto the adjuvant becomesdesorbed. Polyanionic polymers are thought to have the twin propertiesof low pI, and high binding affinity for AlOH that allows smallerconcentrations of the excipient to be used sufficient to prevent the PRPinterference/flocculation problem but insufficient to cause significantor excessive desorption of antigens beneficially adsorbed onto AlOH.

In a particularly preferred embodiment the polyanionic polymer of theinvention is a poly-L-glutamic acid (PLG) homopolymer. Low molecularweight PLG (less than 6000 M_(w), preferably 640-5000) is particularlypreferred (for instance PLG with on average 17 residues with a M_(w) of2178) for optimal clearance from the body post-administration and toensure it does not induce an immune response itself in the host.

PLG is a fully bio-degradable polyamino acid with a pendent freeγ-carboxyl group in each repeat unit (pKa 4.1) and is negatively chargedat a pH7, which renders this homopolymer water-soluble and gives it apolyanionic structure. PLG may be made using conventional peptidesynthesis techniques. It is also available from Sigma-Aldrich in arelatively polydisperse form (e.g. 17 mers with a polydispersity around2.6), or from Neosystem in a relatively monodisperse form (e.g. 8, 16,24 or 32 mers with a polydispersity close to 1). For a preferred 17merof the invention (e.g. from Sigma), PLG is typically present at aconcentration of 290 μg/ml. For a preferred 16mer of the invention (e.g.from Neosystems), PLG is typically present at a concentration of 125-600μg/mL, preferably around 300 or 400 μg/ml, although the skilled personwill appreciate that the above quantities may vary to some degreedepending on the precise composition of the vaccine.

α-PLG is currently used for two main biomedical applications: drugdelivery for cancer therapy (Li et al., Clin. Cancer. Res. 6:2829-2834,2000) and biological glue (Iwata et al., Biomaterials 19:1869-1876,1998). It has not been used as an excipient for intramuscularvaccination.

There are several variables that exist in a vaccine sample which allowthe skilled person to determine the appropriate quantity of polyanionicpolymer (or blend of polyanionic polymers of different M_(w)s) thatshould be used in a vaccine. In general only just enough polyanionicpolymer should be used such that flocculation and/or PRP immuneinterference is prevented. Often the polyanionic polymer is present at ahigher concentration in the vaccine than that of the PRP saccharide.Factors to consider when determining the amount of polyanionic polymerto use are:

1) Charge of the polyanionic polymer—as it becomes more negative, lessconcentration required in the sample. 2) Size of polyanionic polymerchain (on average)—as it increases, less concentration required in thesample. 3) Polydispersity of polyanionic polymer—higher quantities maybe required if uneven bias to low molecular weight species in thesample. 4) Amount of PRP (or saccharide antigen of low pI)—this may befrom 1-20 μg/dose/PS—as amount increases, more polyanionic polymer maybe required. 5) Size of the PRP (or saccharide antigen of low pI)—as thesize increases from oligosaccharide to polysaccharide so may bindingaffinity to AlOH adjuvant (for instance), thereby requiring morepolyanionic polymer. 5) Ratio of PRP (saccharide):carrier (5:1 to 1:5w/w)—as steric hindrance in the conjugate increases polyanionic polymerquantities may need to be adjusted. 6) Charge of adjuvant (with aZPC >8)—as charge increases at same pH, more polyanionic polymer shouldbe required. 7) Size of adjuvant (e.g. AlOH) particle—although it shouldalways be of an injectable size—for a given amount of adjuvant, ifincreased size less polyanionic polymer should be required. 8) Amount ofadjuvant (acceptable range for vaccines being 50-1250 μg per doseAl³⁺)—as amount increases, more polyanionic polymer may be required. 9)Presence of other adsorbed antigens—as a greater amount of Ag isadsorbed on surface of the adjuvant, less polyanionic polymer should berequired.

An assessment of flocculation may be readily carried out be techniquesknown in the art such as sedimentation profile, optical microscopic(Example 2), or even visual observation. In general, vaccines of theinvention comprising adjuvant with a ZPC >8 should be less aggregated(preferably no flocculation observed) than the equivalent formulationwithout polyanionic polymer after 15 minutes of mixing the PRPsaccharide with the vaccine. The conservation of anti-PRP antibodytitres (or reduction of interference to the development of such titres)may be assessed by standard ELISA tests.

Particularly preferred immunogenic compositions of the inventioncomprise PRP (preferably conjugated) and polyanionic polymer that areadvantageously formulated such that the result of multiplying theconcentration of the polyanionic polymer in the composition (in μM) bythe net negative charge of the polyanionic polymer at pH 7.0 divided bythe amount of PRP present in a 0.5 mL dose of the immunogeniccomposition (in μg) is 300-6000, preferably 400-4000, more preferably500-2000, 560-1100, 610-900, 640-800, or 660-700, and most preferablyaround or exactly 680.

The concentration of the polyanionic polymer in the composition shouldagain be measured according to the M_(w) of polyanionic polymer used,and is typically in the range 30-2000 μM, preferably 80-1000, 100-500,150-300, and most preferably around or exactly 200 μM. Alternativelyconcentration may be expressed in units of μg/ml, being typically in therange 45-3000 μg/ml, preferably 120-1500, 150-750, 225-450, and mostpreferably around or exactly 290 or 300 μg/ml.

The net negative charge at pH7.0 of the polyanionic polymer may becalculated by any suitable means. Again this may be an average propertyof the polymer, and should be calculated with respect to the M_(w) ofpolyanionic polymer used. For instance, a PLG polymer with on average 17residues should have a net negative charge of 17. Preferably the netnegative charge should be at least 8, or at least 17, preferably 8-106,10-80, 12-60, 14-40, 16-20, and most preferably around or exactly 17.

It is preferred that the polyanionic polymer of the invention has atleast on average 1 net negative charge at pH 7.0 per 3 monomers,preferably at least 2 per 3 monomers, and most preferably at least onaverage 1 net negative charge for each monomer. The charges may beunevenly arranged over the polymer length, but are preferably evenlyspread over the polymer length.

The immunogenic compositions of the invention typically comprise 1-20,preferably 2.5-10, and most preferably around or exactly 5 μg of PRP(preferably conjugated to carrier protein, the weight of which is notcounted in the above calculations) per 0.5 mL dose. PRP is mostpreferably not intentionally adsorbed onto any adjuvant.

In a highly preferred embodiment an immunogenic composition is providedcomprising 5 μg of PRP conjugated to a carrier protein (preferablytetanus toxoid) and 218 μg of poly-α-L-glutamic acid sodium(approximately 200 μM) per 0.5 mL human dose, wherein the PLG containson average 17 glutamic acid residues (preferably with a M_(w) 2,178, andoptionally with a polydispersity of 2.6).

In all the above immunogenic compositions, further excipients may beadded to those already mentioned. In particular, PRP may be adsorbedonto aluminium phosphate adjuvant (as described in WO 97/00697 and WO99/48525) but is preferably unadsorbed. The immunogenic composition maybe buffered with any suitable buffer that has a pKa that may stabilisethe pH of the composition—typically pH6-7, most preferably pH 6.1. Forexample Histidine buffer may be used, or, preferably, maleate buffer. Ingeneral, buffers (and quantities used) should be selected that do notsignificantly effect the polyanionic polymer's beneficial effect in thecomposition. In general if a buffer is present, less than 10, 5, 4, 3,2, 1, 0.5 or 0.1 mM buffer should be used, preferably around 2 mM.

If the immunogenic composition of the invention is to be lyophilised forstorage purposes, it is preferable that a stabilizing excipient (orcryoprotector) is added to the composition. Any such excipient may beused such as glucose, maltulose, iso-maltulose, lactulose, sucrose,sorbitol, maltose, lactose, iso-maltose, maltitol, lactitol, palatinit,trehalose, raffinose, stachyose, and melezitose, but preferably sucroseis used. Such excipients are typically present in the amount of 1-5%,and preferably around or exactly 2.5% (w/v).

Although the immunogenic compositions of the invention may have anantigenic content consisting of only PRP (preferably conjugated to aprotein carrier), it may comprise one or more further antigens. PRP maybe mixed and stored with these other antigens, or may have them addedextemporaneously (by a practitioner just before administering thecomposition to a patient in need thereof). The polyanionic polymer maybe added to the one or more other antigens before PRP is combined withthem, or, preferably is present with the PRP as a protectant before theother antigens are combined with it. Some of the further antigens may bestored with PRP (preferably lyophilised) and some stored separately(preferably liquid) to be reconstituted together extemporaneously,wherein the polyanionic polymer may be present in either composition,but is preferably present with the PRP.

Preferably, the immunogenic composition in addition to PRP (preferablyconjugated) and polyanionic polymer further comprises one or moremeningococcal capsular oligosaccharide or, preferably,polysaccharide-carrier protein conjugates (see above for preferredcarrier proteins comprising T-helper epitopes, most preferably tetanustoxoid) selected from a group consisting of: MenC, MenY, MenA and MenW(e.g. A+C, A+Y, A+W, C+Y, C+W, Y+W, A+C+Y, A+C+W, A+Y+W, C+Y+W,A+C+Y+W); preferably MenC and/or MenY is included, and most preferablyall 4. These meningococcal components are preferably not intentionallyadsorbed onto any adjuvant. Such immunogenic compositions arebeneficially lyophilised, and may be reconstituted with further antigens(for instance DTPa- or DTPw-based compositions), preferablyextemporaneously. By “extemporaneously” herein it is meant that thevaccine is administered within 1.5 hours or 1 hour of making up thecombined vaccine, preferably within 0.5 hour, most preferably within 15minutes.

Alternatively or in addition to the above meningococcal antigens, theimmunogenic composition may comprise one or more pneumococcal capsularoligosaccharide or polysaccharide-carrier protein conjugates (see abovefor preferred carrier proteins comprising T-helper epitopes, mostpreferably CRM197, diphtheria toxoid, tetanus toxoid or protein D).

Typically pneumococcal capsular oligosaccharides or polysaccharides(preferably the latter) represented in the compositions of the inventioncomprise antigens derived from at least four serotypes of pneumococcus.Preferably the four serotypes comprise 6B, 14, 19F and 23F. Morepreferably, at least 7 serotypes are comprised in the composition, forexample those derived from serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F.More preferably still, at least 11 serotypes are comprised in thecomposition (11 valent), for example those derived from serotypes 1, 3,4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F. In a preferred embodiment of theinvention at least 13 of such conjugated pneumococcal antigens arecomprised, although further antigens, for example 23 valent (such asserotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F,18C, 19A, 19F, 20, 22F, 23F and 33F), are also contemplated by theinvention.

For elderly vaccination (for instance for the prevention of pneumonia)it is advantageous to include serotypes 8 and 12F (and most preferably15 and 22 as well) to the 11 valent pneumococcal antigenic compositiondescribed above to form a 15 valent composition, whereas for infants ortoddlers (where otitis media is of more concern) serotypes 6A and 19Aare advantageously comprised to form a 13 valent composition.

Again such immunogenic compositions comprising pneumococcal antigens arebeneficially lyophilised, and may be reconstituted with further antigens(for instance DTPa-based compositions), preferably extemporaneously.

The immunogenic composition of the invention in addition to PRP andpolyanionic polymer may further comprise one or more (2 or preferablyall 3 [a DTPw or DTPa composition]) further antigens or antigen groupsselected from tetanus toxoid (TT), diphtheria toxoid (DT), andwhole-cell or one or more acellular B. pertussis antigens. The one ormore (2, 3, 4 or all 5) acellular B. pertussis antigens that may becomprised may be selected from the group consisting of: pertussis toxoid(PT), FHA, pertactin (PRN), agglutinogen 2 and agglutinogen 3 (andpreferably comprises the first three).

Such DTPa or DTPw compositions may further comprise either or both ofInactivated Polio Vaccine (IPV) (typically unadsorbed before mixing) andHepatitis B surface antigen (which is preferably adsorbed onto aluminiumphosphate as described in WO 93/24148).

DT, TT, PT, FHA and PRN are well known in the art. The PT component maybe made into a toxoid either chemically or genetically, for example asdescribed in EP 515415. See also EP 427462 and WO 91/12020 for thepreparation of pertussis antigens. Optionally the PT component may berecombinant (for example as described in European Patent Applications EP306318, EP 322533, EP 396964, EP 322115 and EP 275689). Optionally theDT and TT components may also be recombinant. Typically the PT, FHA,PRN, HBsAg (Hepatitis B surface antigen), and PRP components will be inthe range 8-25 μg per 0.5 mL dose of bulk vaccine. The DT, TT, and IPV(inactivated trivalent poliovirus vaccine) components should typicallybe present as approximately 15-25 Lf (flocculating units), 10 Lf, and40/8/32 (type I/II/III) DU respectively per 0.5 mL dose of bulk vaccine.

Suitable components for use in such vaccines are already commerciallyavailable and details may be obtained from the World HealthOrganisation. For example the IPV component may be the Salk inactivatedpolio vaccine. The Hepatitis B surface antigen may comprise the ‘S’antigen as in ENGERIX-B® (SmithKline Beecham Biologicals).

The addition of either lyophilised or liquid PRP (either unadjuvanted oradsorbed onto aluminium phosphate) to a solution of the other componentsof the composition may be performed extemporaneously (see above), orbefore the vaccine leaves the manufacturer. PRP may be combined withpolyanionic polymer before its addition to the other components, or PRPmay be added to other components further comprising polyanionic polymer.

The immunogenic compositions of the invention will typically furthercomprise an adjuvant with a zero point charge greater than 8, 9 or 10,typically an aluminium salt, most often alum or aluminium hydroxide.This will particularly be the case for DTPa-containing compositionswhere one or more DTPa component is preferentially adsorbed ontoaluminium hydroxide. Usually such adjuvant is present in the immunogeniccomposition in the amount of 50-1250 or 100-1000 μg per 0.5 mL dose,usually around or exactly 500 μg per 0.5 mL dose, of which around 50,60, 70, 80, 90 or 95% has antigen (non-PRP, usually one or more DTPaantigens) specifically adsorbed onto its surface.

The presence of such adjuvants would normally tend to flocculate withany PRP present in the composition, however in the immunogeniccompositions of the present invention this is prevented by the presenceof the polyanionic polymer.

Alternatively, and preferably additionally, the presence of thepolyanionic polymer can reduce the immunological interference that theadjuvant has on PRP by over 20, 30, 40, 50, 60, 70, 80, 90, or,preferably, by 100% (interference being measured by taking thedifference in anti-PRP GMT titres in (μg/mL) in an appropriate model[e.g. mouse or rat, or in a well-conducted human clinical trial] betweenadministering a PRP vaccine by itself versus the same PRP vaccine in animmunogenic composition comprising the above adjuvant; the reduction ofinterference being the extent to which the GMT in the immunogeniccomposition is restored to that of the PRP vaccine by itself by theaddition of the polyanionic polymer of the invention to the immunogeniccomposition).

Where the above adjuvant (with a zero-point charge greater than 8, 9, 10or 11) is included in the immunogenic compositions of the invention,this is usually because certain antigens in the composition are mosteffective when adsorbed to the surface of such adjuvants (particularlyaluminium hydroxide).

An advantage of the immunogenic compositions of the invention is thatthe presence of the polyanionic polymer (unlike regular anionic salts)does not cause significant desorption of antigens that are specificallyadsorbed onto the above adjuvant (i.e. with a zero-point charge greaterthan 8). By not causing “significant desorption” it is typically meantthat more than 50, 60, 70, 80 or, preferably, 90% of antigen that hasbeen specifically adsorbed (i.e. intentionally adsorbed and/or adsorbedin a separate adsorption step before mixing with other vaccinecomponents) onto the adjuvant remain adsorbed to the adjuvant after 15minutes or 1 hour of adding the polyanionic polymer to the immunogeniccomposition of the invention. In general it is preferred that in sodoing sufficient antigen remains adsorbed onto the adjuvant in order forthe anti-antigen GMT titre in (μg/mL) in an appropriate model [e.g.mouse or rat, or in a well-conducted human clinical trial] to be morethan 50, 60, 70, 80, 90, or 95% of the GMT titre of the antigen in thesame immunogenic composition without polyanionic polymer in the samemodel.

Typically one or more of the following antigens may be present in theimmunogenic composition of the invention and may have been specifically(and preferably individually) adsorbed onto an adjuvant with a zeropoint charge more than 8 (preferably aluminium hydroxide) before mixingwith the other components of the immunogenic composition of theinvention: diphtheria toxoid, tetanus toxoid, pertussis toxoid, FHA andpertactin (PRN). Preferably at least PRN is adsorbed onto aluminiumhydroxide, and most preferably all 5 of the components are adsorbed ontoaluminium hydroxide.

Methods of adsorbing DTPa and DTPw antigens onto aluminium adjuvants areknown in the art. See for example WO 93/24148 and WO 97/00697. Usuallycomponents adsorbed onto adjuvant are left for a period of at least 10minutes at room temperature at an appropriate pH for adsorbing most andpreferably all of the antigen before mixing the antigens together in thecombination immunogenic compositions of the present invention.

Other components are preferably unadsorbed (such as IPV) or adsorbedspecifically onto other adjuvants—Hepatitis B surface antigen (HepBsa)being preferably adsorbed onto aluminium phosphate (as described in WO93/24148) before mixing with other components.

Typical combination vaccines of the invention comprise: DTPa IPV HepBsaPRP (preferably conjugated to TT) PLG; DTPa IPV HepBsa PLG PRP MenCand/or MenY (where the capsular saccharides are preferably conjugated toTT). As mentioned above, PLG may reduce aggregation events which reducethe immunogenicity of meningococcal capsular saccharide epitopes incertain vaccines. Such combinations of the invention may comprise: DTPwHepBsa PLG Hib MenA and/or MenC (wherein the capsular saccharides arepreferably conjugated to TT).

In a preferred embodiment, a vaccine is provided comprising theimmunogenic composition of the invention and a pharmaceuticallyacceptable excipient. The pH of the vaccines of the present invention isusually between pH6-7, preferably pH 6.1.

Vaccine preparation is generally described in Vaccine Design—The Subunitand adjuvant approach Ed Powell and Newman; Pellum Press. Advantageouslythe combination vaccine according to the invention is a paediatricvaccine.

The amount of polysaccharide or oligosaccharide conjugate antigen ineach vaccine dose is selected as an amount which induces animmunoprotective response without significant, adverse side effects intypical vaccinees. Such amount will vary depending on which specificimmunogens are employed. Generally it is expected that each dose willcomprise 1-1000 μg of conjugated polysaccharide or oligosaccharide(expressed in amount of saccharide), preferably 2-100 μg, morepreferably 4-40, 2-15, or 3-10 μg, most preferably around or exactly 5μg.

The content of protein antigens in the vaccine will typically be in therange 1-100 μg, preferably 5-50 μg, most typically in the range 5-25 μg.

An optimal amount of antigen for a particular vaccine can be ascertainedby standard studies involving observation of antibody titres and otherresponses in subjects. Following an initial vaccination, subjects mayreceive one or two booster injections at about 4 weeks intervals orlonger.

The vaccine preparations of the present invention may be used to protector treat a mammal (preferably human) susceptible to infection, by meansof administering said vaccine via systemic or mucosal route. Theseadministrations may include injection via the intramuscular,intraperitoneal, intradermal or subcutaneous routes; or (less preferred)via mucosal administration to the oral/alimentary, respiratory,genitourinary tracts.

There is further provided a method of preventing or treating H.influenzae B disease (or disease associated with antigen of low pI ofthe invention) by administering a pharmaceutically effective amount ofthe vaccine of the invention to a patient in need thereof, and a use ofthe immunogenic composition or vaccine of the invention in themanufacture of a medicament for the prevention or treatment of H.influenzae B disease.

The present invention additionally provides a method for reducing theimmunological interference and/or flocculation of a Haemophilusinfluenzae B capsular polysaccharide or oligosaccharide (PRP), which ispreferably conjugated, in a combination vaccine (a vaccine of theinvention comprising PRP and at least one further antigen) comprisingone or more further antigens adsorbed to an adjuvant with a zero pointcharge greater than 8 (as described above, preferably aluminiumhydroxide), wherein such method comprises the steps of:

-   -   (i) adsorbing the one or more further antigens onto the        adjuvant;    -   (ii) adding a polyanionic polymer to said one or more further        antigens; and    -   (iii) then adding an immunogenic composition comprising PRP to        said one or more further antigens;        or comprising the steps of:    -   (i) adsorbing the one or more further antigens onto the        adjuvant; and    -   (ii) adding an immunogenic composition of the invention        comprising PRP and a polyanionic polymer to said one or more        further antigens.

In the former the adjuvant is policed before PRP is added, in the latterPRP is protected before it is added to the adjuvant. Preferably, ineither method the components are mixed extemporaneously. The immungeniccomposition comprising PRP is preferably lyophilised for greateststability, most preferably in the presence of a stabilizing excipient(as described above). The immungenic composition comprising PRP ispreferably combined with one or more conjugated meningococcal capsularoligosaccharides or polysaccharides and/or one or more conjugatedpneumococcal capsular oligosaccharides or polysaccharides (as describedabove).

Preferably the one or more further antigens adsorbed to adjuvant areselected from diphtheria toxoid, tetanus toxoid, pertussis toxoid, FHAand pertactin, most preferably all, as described above. Most preferably,as described above, the presence of the polyanionic polymer in thecombination vaccine does not cause significant desorption of the one ormore further antigens adsorbed to the adjuvant.

Further provided is a use of a polyanionic polymer (as described above)in an immunogenic composition further comprising a Haemophilusinfluenzae B capsular polysaccharide or oligosaccharide (PRP),preferably conjugated, as a means for protecting the immune response ofPRP. By protecting the immune response it is meant retaining more than50, 60, 70, 80, 90, or 95% of the anti-PRP GMT titre of the PRPcomponent by itself, regardless of whether the immunogenic compositionis later combined with a vaccine comprising adjuvant with a zero-pointcharge greater than 8 (as described above).

A kit is further provided comprising: i) a first immunogenic compositioncomprising a Haemophilus influenzae B capsular polysaccharide oroligosaccharide (PRP), preferably conjugated, and a polyanionic polymer(as described above); and ii) a second immunogenic compositioncomprising one or more antigens adsorbed onto an adjuvant with a zeropoint charge greater than 8 (preferably aluminium hydroxide).Preferably, the first immunogenic composition is lyophilised and furthercomprises a stabilizing excipient (as described above), preferablysucrose, and the second immunogenic composition is liquid. It isenvisaged the contents of the kit may be simply administered byextemporaneously reconstituting the first immunogenic composition withthe second immunogenic composition, and administering the resultingmixed composition. It is highly preferred that the polyanionic polymersof the present invention can dissolve in aqueous solution faster thanPRP or PRP conjugates so that, when co-lyophilised, the polymer (such asPLG) may effectively protect the slower dissolving PRP when it isreconstituted in a liquid composition comprising an adjuvant with a zeropoint charge greater than 8.

Preferably the first immunogenic composition further comprises one ormore conjugated meningococcal capsular oligosaccharides orpolysaccharides selected from a group consisting of: MenC, MenY, MenAand MenW (e.g. A+C, A+Y, A+W, C+Y, C+W, Y+W, A+C+Y, A+C+W, A+Y+W, C+Y+W,A+C+Y+W), preferably MenC and/or MenY, and/or one or more conjugatedpneumococcal capsular oligosaccharides or polysaccharides (as describedabove). Preferably, the second immunogenic composition comprises one ormore (most preferably all) antigens selected from a group consisting of:diphtheria toxoid, tetanus toxoid, pertussis toxoid, FHA and pertactin.It may alternatively comprise DTPw.

As described above, all embodiments herein concerning PRP may equally(and alternatively) extend to other antigens or saccharides of low pI,whilst still taking advantage of the benefits of the invention (forinstance the reduction or prevention of flocculation/aggregationevents).

Further provided is the use of a polyanionic polymer of the invention inthe manufacture of an immunogenic composition for the prevention ofaggregation or flocculation occurring in said composition. Preferablythe immunogenic composition is an immunogenic composition of theinvention, e.g. one as hereinbefore described.

The following examples illustrate, but do not the limit the scope of,the invention.

EXAMPLES Example 1

Numerous experiments were done with PRP polysaccharide conjugated totetanus toxoid (PRP-T) in conjunction with PLG (Hib-PLG) experimentallots. The following parameters were evaluated:

-   -   the molecular weight and content of PLG,    -   the content of PRP-T,    -   the stabiliser for the lyophilisation.

The following results are from the in-vitro, pre-clinical and potencytesting of these experimental lots.

Results with the Experimental LotsIn-Vitro Data with Hib-PLG

Three types of mixing steps were followed to demonstrate the efficacy ofPLG in reducing the physical interaction between PRP-T and Al(OH)₃,including mixing the commercially available INFANRIX PENTA™(DTPaHepBIPV) vaccine with Hib-PLG vaccine:

-   -   Step 1: pre-saturation whereby PLG was first adsorbed on        Al(OH)₃, then PRP-T was added.    -   Step 2: competition whereby PLG was put in competition with        PRP-T for adsorption on Al(OH)₃    -   INFANRIX PENTA™ Hib-PLG: whereby PLG and PRP-T were        co-lyophilized and then put in competition for adsorption in        INFANRIX PENTA™ vaccine, containing 500 μg Al(OH)₃

In all 3 steps, PLG was able to avoid flocculation induced by PRP-T (seeFIGS. 1 and 2).

The 200 μM PLG (Mw 2,200) content was selected for clinical formulationas:

-   -   no flocculation was observed,    -   close to 80% PRP-T was non-adsorbed (according to the Dionex        test)    -   PLG is fully adsorbed    -   all major components of INFANRIX® (DT, TT, PT, FHA, PRN [or        69K], IPV and HB surface antigen) were not affected.    -   Both lactose and sucrose were found to be efficient        cryoprotectors.

In FIG. 1 pre-saturation of Al(OH)₃ with PLG (106 residues) is shown. 10μg PRP/dose, 500 μg AlOH. It was also found that 2000 uM PLG (Mw 1043-8residues) could keep 80% of PRP-T in the supernatant (10 μg PRP/dose,500 μg AlOH) with no flocculation resulting.

FIG. 2 shows the competition between PRP-T and PLG (85 residues). 100 μMPLG Mw 10,900 are able to limit adsorption of 10 μg Hib on 50 (to mimichypothetical free Al(OH)₃ in INFANRIX PENTA™) as well as on 500 μgAl(OH)₃ (=full Al(OH)₃ dose in INFANRIX PENTA™).

In addition, 500 μm PLG Mw 2,178 (17 residues) are able to limitadsorption of 10 μg Hib on Al(OH)₃ after reconstitution of [Hib-PLG]cake with INFANRIX PENTA™ (1 h contact, then centrifugation 6 min 6500 gand dosage of Hib in supernatant by ELISA PRR′P-TT or Dionex dosage), ascan 75 μM PLG Mw 10,900 (85 residues).

PLG Mw 2,178 (17 residues) is able to limit adsorption of 5 μg Hib(PRP-T) on Al(OH)₃ after reconstitution of [Hib-PLG] cake with INFANRIXPENTA™ (1 h contact, then centrifugation 6 min 6500 g and dosage of Hibin supernatant by ELISA PRR′P-TT or Dionex dosage)—175 and 200 μM wereoptimal concentrations in that there is an absence of flocculation andINFANRIX® antigen adsorption is retained.

PLG Mw 10,800 (85 residues) is able to limit adsorption of 5 μg Hib(amount of PRP in PRP-T) on Al(OH)₃ after reconstitution of [Hib-PLG]cake with INFANRIX PENTA™ (1 h contact, then centrifugation 6 min 6500 gand dosage of Hib in supernatant by ELISA PRR′P-TT or Dionex dosage)—30and 35 μM were optimal concentrations in that there is an absence offlocculation and INFANRIX® antigen adsorption is retained.

Pre-Clinical Immunogenicity Data with Hib-PLG

Hib-PLG experimental formulations were evaluated in a rabbit model ofimmunogenicity and a baby rat model allowing evaluation of the Hib(PRP-T conjugate) immune interference induced by combination of INFANRIXPENTA™ a and Hib vaccines. Moreover, impact of Hib-PLG on the efficacyof INFANRIX PENTA™ was evaluated in a B. pertussis lung colonizationmurine model.

Rabbit Model of Immunogenicity Study Design

In this model, 5 week old New Zealand female rabbits wereintramuscularly immunised three times at two weeks intervals (day 0, 14,28) with ¼ of a vaccine human dose. A sample size of 10 animals pergroup was used. Blood samples were taken on day 21, 28, 35 and 42.Anti-PRP antibodies were measure by ELISA and are expressed in μg/ml.

Vaccines Administered

Formulations of PRP-T (5 μg PRP) containing high (10900 MW) and low(2200 MW) molecular weight PLG were evaluated in two concentrations. Asimilar Hib formulation but without PLG was included as control. Seebelow for details of vaccines administered by group.

Group Vaccine 1 Hib (5 μg) PLG HMW (10900) 30 μM 2 Hib (5 μg) PLG HMW(10900) 70 μM 3 Hib (5 μg) PLG LMW (2200) 175 μM 4 Hib (5 μg) PLG LMW(2200) 450 μM 5 Hib (5 μg) 6 PLG HMW (10900) 40μ

Results See FIG. 3.

Although some variability of PRP response can be observed in this rabbitmodel, no significant difference was demonstrated between groups.

There was a slight reduction of immunogenicity observed in rabbitshaving received Hib-PLG L Mw 175 μM, but all other formulations inducedanti-PRP antibody levels similar to the Hib control group withoutaddition of PLG.

No induction of anti-PLG antibodies after immunization of rabbits withthese Hib-PLG experimental formulations were demonstrated.

Baby Rat Model of Hib Interference Study Design

Seven day old OFA rats were intramuscularly immunised three times at twoweeks intervals (day 0, 14, 28) with 1/10 of a vaccine human 0.5 mLdose. An equal repartition of male and female rats was realised. Asample size of 20 animals per group was used. Blood samples wererealised on day 35 and anti-PRP antibodies were measured by ELISA andexpressed in μg/ml.

Vaccines Administered

As a control of interference in the baby rat model, Hib combined withINFANRIX PENTA™ (10 μg PRP) was administered as well as Hib (10 μg)co-administered with INFANRIX PENTA™.

Hib (5 μg) formulated alone or containing various amounts of PLG wereevaluated after reconstitution with INFANRIX PENTA™.

See below for details of vaccines administered by group

Group Vaccine 1 Hib (10 μg) + INFANRIX PENTA ™ 2 Hib (10 μg)reconstituted with INFANRIX PENTA ™ 3 Hib (5 μg) PLG HMW (10900) 30 μMreconstituted with INFANRIX PENTA ™ 4 Hib (5 μg) PLG HMW (10900) 75 μMreconstituted with INFANRIX PENTA ™ 5 Hib (5 μg) PLG LMW (2200) 175 μMreconstituted with INFANRIX PENTA ™ 6 Hib (5 μg) PLG LMW (2200) 500 μMreconstituted with INFANRIX PENTA ™ 7 Hib (5 μg) reconstituted withINFANRIX PENTA ™

Results See FIG. 4.

When Hib is administered with INFANRIX PENTA™ immune interference wasobserved as compared with Hib co-administered separately with INFANRIXPENTA™.

The presence of PLG in Hib formulations resulted in partial or totalrestoration of the anti-Hib response. Indeed, a higher immune responseagainst PRP was observed in all Hib-formulations containing PLG comparedto the control group (Group 7).

These results demonstrate that prevention of Hib interaction withadsorption on Al(OH)₃ by addition of PLG can restore high anti-PRPantibody titres elicited by monovalent Hib vaccine.

No induction of anti-PLG antibodies after immunization of baby rats withthese Hib-PLG experimental formulations were demonstrated.

Maintenance of Adsorption of Antigens on Aluminium Hydroxide

5 μg (saccharide) of PRP-T (Hib) was combined with different amounts ofPLG (Sigma) of Mw 2,600 to realise either 0, 175 or 200 μM finalconcentration in the reconstituted vaccine. The samples were lyophilisedin the presence of sucrose. The samples were then reconstituted withINFANRIX PENTA™ and after 1 hour the recovery of unadsorbed antigen inthe supernatant (s/n) was measured. The results were as below:

Control (0 PLG) PLG 175 μM PLG 200 μM Flocculation? Yes No No % Hib ins/n 0 0 0 % DT in s/n 0 1 4 % TT in s/n 0 8 17 % PT in s/n 3 3 3 % FHAin s/n 0 0 0 % 69K (PRN) in s/n 0 11 15 % HepBsa in s/n 1 1 1 % IPV typeI in s/n 5 5 17 % IPV type II in s/n 5 5 5 % IPV type III in s/n 5 38 54

Conclusion:

Antigens that should be beneficially adsorbed to AlOH adjuvant (TT andpertactin) are maintained to a great extent in an adsorbed state. IPVtype III seems most sensitive to desorption, however it is adsorbed tosome degree. Also it should be noted IPV antigens are not specificallyadsorbed to adjuvant before combination with the other antigens; ratherIPV is mixed with the other antigens in an unadsorbed state.Interestingly in this experiment Hib seems to be fully adsorbed onto theadjuvant, but the PLG still prevents flocculation events (and Hib immuneinterference) from occurring. Typically the adjuvant is not saturatedwith PLG at 200 μM (290 μg/ml), and becomes saturated (free PLGbeginning to be present in solution) only at concentrations of around650 μg/ml at which point the PRP is around 60% free in solution. The PLGvaccines pass potency QC release criteria including pertussis challengetests.

Example 2 Analysis of [DTPw/MenACHib] combinations by Optical Microscopy

The reconstitution of DTPaHepB IPV with Hib conjugate inducesflocculation of the aluminium contained in DTPaHepB IPV (see FIG. 5).These may be observed visually and by optical microscopy, and may bemeasured by size and sedimentation analysis. FIG. 7 shows an opticalmicroscope picture of INFANRIX PENTA™ (DTPaHepBIPV). FIG. 8 shows thesample flocculating when 5 μg saccharide of PRP-TT is added to thesample. FIG. 9 shows that no flocculation occurs in the presence of 200μM PLG Mw 2,200 (Sigma).

This example sought to discover whether MenACHib-TT polysaccharideconjugates induce the apparition of aluminium flocculation when mixedwith DTPwHB vaccines, and whether PLG could alleviate this.

Methods and Materials

The DTPaHepB IPV (INFANRIX®) tested had 500 μg Al(OH)₃, 200 μg AlPO₄ perhuman dose. The DTPwHepB (TRITANRIX™) tested had 260 μg Al(OH)₃, 370 μgAlPO₄ per human dose. PRP-TT (Hib) was unadjuvanted, as were the MenA-TTand MenC-TT capsular polysaccharide conjugates. 5 μg saccharide of theconjugates per dose were used in all experiments.

Analysis was by optical microscopy coupled to an image analyzer (KS400system). DTPw samples were mixed with MenACHib samples comprising PLGfor 15 minutes before observation.

Results

Flocculations were not observed in DTPw controls (FIG. 10) but wereobserved in DTPw samples with reconstituted MenACHib-TT (FIG. 11).MenACHib-TT alone does not flocculate.

Electrostatic interactions between polyanionic MenA, MenC and Hibpolysaccharide conjugates and cationic Al(OH)₃ are probably the reasonfor this phenomenon (FIG. 5).

Flocculation may be reduced by the addition of a competitive polyanionicexcipient e.g. PLG. FIG. 12 shows that DTPwHepB/MenACHib with 250 μM PLGexhibits reduced flocculation.

Conclusions

MenACHib-TT conjugates can induce aluminium flocculation in[DTPw/MenACHib] combinations. This may be reduced by the addition of acompetitive polyanionic excipient.

Example 3 A Phase II, Randomized, Partially Blinded Clinical Trial toEvaluate the Immunogenicity and Reactogenicity of GSK Biologicals'Combined DTPa-HBV-IPV/Hib Vaccine (New Formulation with PLG) as Comparedto GSK Biologicals' Licensed DTPa-HBV-IPV/Hib Vaccine (INFANRIX HEXA™)and to the Concomitant Administration of GSK Biologicals' LicensedDTPa-HBV-IPV (INFANRIX PENTA™) and Hib (HIBERIX™®) Vaccines, when Givenas a Primary Vaccination to Healthy Infants at 2, 3 and 4 Months of Age

The study was conducted at 9 centres in Poland.

Objectives: Primary:

To evaluate the immunogenicity of DTPa-HBV-IPV/Hib (new formulation withPLG) in terms of anti-PRP antibody response in comparison with Hib(HIBERIX™®) co-administered with DTPa-HBV-IPV (INFANRIX PENTA™) and incomparison with DTPa-HBV-IPV/Hib (INFANRIX HEXA™, licensed formulation)one month after a three-dose primary vaccination course.

Secondary:

To evaluate the immunogenicity of DTPa-HBV-IPV/Hib (new formulation withPLG) in terms of antibody response to all other vaccine antigens incomparison with Hib co-administered with DTPa-HBV-IPV and in comparisonwith DTPa-HBV-IPV/Hib (licensed formulation) one month after athree-dose primary vaccination course.

To evaluate the immune response to Poly L Glutamate (PLG) in terms ofantibody detection and concentration one month after a three-doseprimary vaccination course.

To assess the safety and reactogenicity of the study vaccines in termsof solicited symptoms, unsolicited symptoms and serious adverse events(SAEs).

Study Design:

Partially blinded, randomized study with three parallel groups:

-   -   DTPa-HBV-IPV/Hib (new formulation with PLG)    -   DTPa-HBV-IPV/Hib (licensed formulation)    -   DTPa-HBV-IPV+Hib (licensed formulations)        The study was conducted in a single-blind manner with respect to        the groups receiving DTPa-HBV-IPV/Hib (new formulation with PLG)        and DTPa-HBV-IPV/Hib (licensed formulation). The group who        received two separate injections (DTPa-HBV-IPV+Hib) was open.        Three doses of each vaccine were administered intramuscularly        according to a 0, 1-month schedule to healthy infants who were 2        months of age at the time of the first dose (FIG. 6). Two blood        samples (at 2 and 5 months of age) were taken from each subject.

Number of Subjects:

Completed: 149 (49 in group DTPa-HBV-IPV/Hib (new formulation), 50 ingroup DTPa-HBV-IPV/Hib (licensed formulation), 50 in groupDTPa-HBV-IPV+Hib)Safety: Total vaccinated cohort: 150 (49 in group DTPa-HBV-IPV/Hib (newformulation), 50 in group DTPa-HBV-IPV/Hib (licensed formulation), 51 ingroup DTPa-HBV-IPV+Hib)Immunogenicity: ATP Cohort: 147 (48 in group DTPa-HBV-IPV/Hib (newformulation), 49 in group DTPa-HBV-IPV/Hib (licensed formulation), 50 ingroup DTPa-HBV-IPV+Hib)

Diagnosis and Criteria for Inclusion:

Infants born after a normal gestation period of 36-42 weeks, 8-12 weeksof age at the time of the first vaccine dose, free of obvious healthproblems as established by medical history and clinical examinationbefore study entry, and with written informed consent obtained fromparents or guardians of the subject.

Study Vaccine, Dose, Mode of Administration: Vaccination Schedule/Site:

Each of the study vaccines was administered at 2, 3 and 4 months of age.The vaccines were administered as a single intramuscular injection inthe left thigh for the Haemophilus influenzae type b vaccine and theright thigh for all other vaccines.

To prepare the DTPa-HBV-IPV/Hib vaccine (new formulation and licensedformulation), the liquid DTPa-HBV-IPV vaccine was reconstituted with therespective Hib vaccine. The Haemophilus influenzae type b vaccine wasreconstituted with the supplied saline diluent. Vaccinecomposition/dose:

-   -   One dose of GSK Biologicals' DTPa-HBV-IPV/Hib vaccine (new        formulation) consisted of: DTPa-HBV-IPV component:

Diphtheria toxoid ≧30 IU (25 Lf), tetanus toxoid ≧40 IU (10 Lf),pertussis toxoid (PT) 25 μg, filamentous hemagglutinin (FHA) 25 μg,pertactin (PRN) 8 μg, hepatitis B surface antigen (recombinant) 10 μg,polioviruses type 1: 40 D antigen units, type 2: 8 D antigen units, type3: 32 D antigen units, aluminium salts 0.7 mg, 2-phenoxyethanol ≦2.5 mg,sodium chloride 150 mM, formalin ≦100 μg, neomycin sulphate ≦25 μg andpolysorbate ≦50 μg.

Hib Component:

Polyribosylribitol phosphate (PRP) 5 μg, tetanus toxoid 10-20 μg,saccharose 2.52% and poly L glutamate 200 μM [290 μg/ml] (Mw 2,200, 17residues).

-   -   One dose of GSK Biologicals' DTPa-HBV-IPV/Hib vaccine (licensed        formulation) consisted of:

DTPa-HBV-IPV Component:

Diphtheria toxoid ≧30 IU (25 Lf), tetanus toxoid ≧40 IU (10 Lf),pertussis toxoid (PT) 25 μg, filamentous hemagglutinin (FHA) 25 μg,pertactin (PRN) 8 μg, hepatitis B surface antigen (recombinant) 10 μg,polioviruses type 1: 40 D antigen units, type 2: 8 D antigen units, type3: 32 D antigen units, aluminium salts 0.7 mg, 2-phenoxyethanol ≦2.5 mg,sodium chloride 150 mM, formalin ≦100 μg, neomycin sulphate ≦25 μg andpolysorbate ≦50 μg.

Hib Component:

Polyribosylribitol phosphate (PRP) 10 μg, tetanus toxoid 20-40 μg,aluminium 0.12 mg and lactose 12.6 mg.

One dose of GSK Biologicals' DTPa-HBV-IPV vaccine consisted of:

Diphtheria toxoid ≧30 IU (25 Lf), tetanus toxoid ≧40 IU (10 Lf),pertussis toxoid (PT) 25 μg, filamentous hemagglutinin (FHA) 25 μg,pertactin (PRN) 8 μg, hepatitis B surface antigen (recombinant) 10 μg,polioviruses type 1: 40 D antigen units, type 2: 8 D antigen units, type3: 32 D antigen units, aluminium salts 0.7 mg, 2-phenoxyethanol ≦2.5 mg,sodium chloride 150 mM, formalin ≦100 μg, neomycin sulphate ≦25 μg andpolysorbate ≦50 μg.

One dose of GSK Biologicals' Haemophilus influenzae type b vaccineconsisted of: Polyribosylribitol phosphate (PRP) 10 μg, tetanus toxoid20-40 μg and lactose 10.08 mg.

Criteria for Evaluation: Immunogenicity: Primary Endpoint

Anti-PRP antibody concentrations one month after the three dose primaryvaccination course.

Secondary Endpoint Immunogenicity:

One month after the three-dose primary vaccination course,

-   -   Seroprotection status for the vaccine antigens were as follows:

Anti-PRP antibody concentrations ≧0.15 μg/ml and ≧1.0 μg/ml

Anti-HBs antibody concentrations ≧10 mIU/ml

Anti-diphtheria toxoid antibody concentrations ≧0.1 IU/ml

Anti-tetanus toxoid antibody concentrations ≧0.1 IU/ml

Anti-poliovirus type 1 antibody titres ≧8

Anti-poliovirus type 2 antibody titres ≧8

Anti-poliovirus type 3 antibody titres ≧8

-   -   Anti-PT, anti-FHA and anti-PRN seropositivity rates (antibody        concentrations ≧5 EL.U/ml).    -   Anti-PT, anti-FHA, anti-PRN, anti-diphtheria and anti-tetanus        toxoids, anti-poliovirus types 1, 2 and 3, and anti-HBs antibody        concentrations.    -   Vaccine response rates to PT, FHA and PRN, defined as appearance        of antibodies in subjects who were seronegative at        pre-vaccination time point (i.e. with antibody concentrations <5        EL.U/ml) or at least maintenance of pre-vaccination antibody        concentrations in subjects who were seropositive at        pre-vaccination time point (i.e. with antibody concentrations ≧5        EL.U/ml), taking into consideration the decreasing maternal        antibodies.    -   Anti-PLG antibody detection and concentration.

Safety: Recording of solicited symptoms (pain, redness, swelling,drowsiness, fever, irritability/fussiness, loss of appetite) during the4-day follow-up period (Day 0-Day 3) after each study vaccination.Recording of unsolicited symptoms occurring from Day 0 to Day 30 aftereach study vaccination and serious adverse events (SAE) during theentire study period.

-   -   Occurrence of solicited local symptoms during the 4-day        follow-up period (Day 0-Day 3) after vaccination.    -   Occurrence of solicited general symptoms during the 4-day        follow-up period (Day 0-Day 3) after vaccination.    -   Occurrence of unsolicited symptoms during the 31-day follow-up        period (Day 0-Day 30) after vaccination.

Occurrence of serious adverse events (SAEs) during the entire study.

Statistical Methods:

Analysis of immunogenicity: The immunogenicity analysis was performed onthe ATP cohort. The following analyses has been performed for eachtreatment group at each time-point a serological result was available:Geometric mean antibody concentrations/titers (GMCs/GMTs) with 95%confidence intervals (CIs) were tabulated for anti-PRP and forantibodies against each vaccine antigen. Seropositivity/seroprotectionrates with exact 95% CIs were tabulated. Vaccine response rates(post-dose 3) to pertussis antigens and their exact 95% CI was tabulatedand the distribution of antibody concentrations/titers for each antigenpost-dose 3 was displayed using reverse cumulative distribution curves.

The immunogenicity of the DTPa-HBV-IPV/Hib (new formulation with PLG)vaccine was compared with that of the licensed vaccines by computingboth the 95% CI for the group GMC/GMT ratio for each antigen one monthafter the third vaccine dose and the standardized asymptotic 95% CI forthe difference in seroprotection/seropositivity rates for each antigenone month after the third vaccine dose.

Analysis of safety: The safety analysis was performed on the Totalvaccinated cohort. Within groups, the incidence of each solicitedsymptom that occurred within 4 days after vaccination was determined pergroup by tabulating the percentage of doses with the symptom and itsexact 95% CI both at each dose and over the three doses for all groupsand by tabulating the percentage of subjects with the symptom and itsexact 95% CI for all groups. The percentage of subjects with unsolicitedsymptoms with its exact 95% CI was tabulated by group and by WHOpreferred term and similar tabulation was done for grade “3” unsolicitedsymptoms and for unsolicited symptoms possible related to vaccination.In addition, the number of subjects who experienced at least one SeriousAdverse Event (SAE) during the entire study period was reported. Atwo-sided p-value from the Fisher exact test and the standardizedasymptotic 95% CIs for the differences between DTPa-HBV-IPV/Hib (newformulation) and each of the control groups were computed for thepercentage of subjects with a given symptom of any intensity within 4days after vaccination as well as for the percentage of subjects withrectal fever >39.0° C. within 4 days after vaccination and thepercentage of subjects who received antipyretics within 4 days aftervaccination.

Immunogenicity Results:

The immune response to PRP after vaccination with DTPa-HBV-IPV/Hib PLGwas similar to that to HIBERIX® (HIB vaccine) and statistically higherthan the response to INFANRIX HEXA™ (DTPa-HBV-IPV/Hib vaccine) (p<0.05),both in terms of GMCs and of seroprotection rates (Table 1).

The immune response to all the antigen components of the vaccine weresimilar in terms of seroprotection rates, for diphtheria, tetanus,hepatitis an poliovirus and in terms vaccine response rates to pertussisantigens (Table 2).

TABLE 1 response to the PRP antigen in the three study groups ((ATPcohort for immunogenicity) ≧0.15 μg/ml ≧1 μg/ml GMC (μg/ml) 95% CI 95%CI 95% CI Group Timing N n % LL UL n % LL UL Value LL ULDTPa-HBV-IPV/HibPLG PRE 48 17 35.4 22.2 50.5 1 2.1 0.1 11.1 0.137 0.1060.179 PIII 47 47 100.0 92.5 100.0 36 76.6 62.0 87.7 3.375 2.314 4.924DTPa-HBV-IPV/Hib PRE 49 18 36.7 23.4 51.7 6 12.2 4.6 24.8 0.177 0.1220.255 PIII 49 42 85.7 72.8 94.1 29 59.2 44.2 73.0 1.164 0.711 1.904DTPa-HBV-IPV + PRE 50 17 34.0 21.2 48.8 4 8.0 2.2 19.2 0.153 0.112 0.209HIBERIX ® PIII 50 48 96.0 86.3 99.5 36 72.0 57.5 83.8 3.014 1.846 4.920N: number of subjects with available results %: percentage of subjectswith anti-PRP concentration above the specified cut-off 95% CI; LL, UL:95% confidence interval; lower and upper limit

TABLE 2 Percentage of subjects seroprotected and vaccine response topertussis antigens after primart vaccination (ATP cohort forimmunogenicity) DTPa- DTPa-HBV- DTPa-HBV- HBV-IPV + IPV/HibPLG IPV/HibHIBERIX ® 95% CI 95% CI 95% CI N % LL UL N % LL UL N % LL ULSeroprotection Anti-D (≧0.1 IU/ml) 48 97.9 88.9 99.9 49 100 92.7 100 50100 92.9 100 Anti-T (≧0.1 IU/ml) 47 100 92.5 100 49 100 92.7 100 50 10092.9 100 Anti-HBs (≧10 mIU/ml) 47 100 92.5 100 49 100 92.7 100 50 98.089.4 99.9 Anti-polio 1 (≧8) 33 100 89.4 100 41 100 91.4 100 41 100 91.4100 Anti-polio 2 (≧8) 34 100 89.7 100 39 97.4 86.5 99.9 36 100 90.3 100Anti-polio 3 (≧8) 32 100 89.1 100 34 100 89.7 100 33 100 89.4 100Vaccine response Anti-PT 48 100 92.6 100 48 97.9 88.9 99.9 48 93.8 82.898.7 Anti-FHA 48 89.6 77.3 96.5 49 93.9 83.1 98.7 50 90.0 78.2 96.7Anti-PRN 48 91.7 80 97.7 49 95.9 86 99.5 50 90.0 78.2 96.7 N: number ofsubjects with available results %: percentage of subjects seroprotectedor with vaccine response 95% CI; LL, UL: 95% confidence interval; lowerand upper limit

Safety Results: The incidences of any local or general symptom werecomparable between the three study groups. Incidences were not increasedin the group receiving DTPa-HBV-IPV/HibPLG as compared with the otherthree study groups. Fever (rectal temperature) above 39.5° C. was notreported in any of the groups. Fever (rectal temperature) above 39.0° C.was not reported in the DTPa-HBV-IPV/HibPLG group.

TABLE 3 Safety results for the ATP safety cohort. DTPa-HBV- DTPa-HBV-DTPa-HBV- IPV + IPV/HibPLG IPV/Hib HIBERIX ® (N = 49) (N = 50) (N = 51)Intensity n % 95% CI n % 95% CI n % 95% CI Solicited local symptoms PainAny 19 38.8 25.2 53.8 17 34.0 21.2 48.8 20 39.2 25.8 53.9 Grade “3” 00.0 0.0 7.3 1 2.0 0.1 10.6 2 3.9 0.5 13.5 Redness Any 27 55.1 40.2 69.326 52.0 37.4 66.3 23 45.1 31.1 59.7 >20 mm 1 2.0 0.1 10.9 4 8.0 2.2 19.24 7.8 2.2 18.9 Swelling Any 20 40.8 27.0 55.8 17 34.0 21.2 48.8 20 39.225.8 53.9 >20 mm 6 12.2 4.6 24.8 5 10.0 3.3 21.8 11 21.6 11.3 35.3Solicited general symptoms: Drowsiness Any 22 44.9 30.7 59.8 26 52.037.4 66.3 26 51.0 36.6 65.2 Grade “3” 1 2.0 0.1 10.9 1 2.0 0.1 10.6 00.0 0.0 7.0 Related 22 44.9 30.7 59.8 24 48.0 33.7 62.6 24 47.1 32.961.5 Irritability Any 29 59.2 44.2 73.0 30 60.0 45.2 73.6 30 58.8 44.272.4 Grade “3” 4 8.2 2.3 19.6 1 2.0 0.1 10.6 3 5.9 1.2 16.2 Related 2857.1 42.2 71.2 28 56.0 41.3 70.0 29 56.9 42.2 70.7 Loss of appetite Any17 34.7 21.7 49.6 16 32.0 19.5 46.7 14 27.5 15.9 41.7 Grade “3” 0 0.00.0 7.3 0 0.0 0.0 7.1 0 0.0 0.0 7.0 Related 17 34.7 21.7 49.6 13 26.014.6 40.3 12 23.5 12.8 37.5 Fever ≧38° C. 7 14.3 5.9 27.2 8 16.0 7.229.1 8 15.7 7.0 28.6 >38.5° C. 2 10.5 3.4 22.2 8 16.0 7.2 29.1 7 13.75.7 26.3 >39° C. 0 0.0 0.0 7.3 0 0.0 0.0 7.1 2 3.9 0.5 13.5 >39.5° C. 00.0 0.0 7.3 0 0.0 0.0 7.1 0 0.0 0.0 7.0 Related 5 10.2 3.4 22.2 8 16.07.2 29.1 7 13.7 5.7 26.3

Serious Adverse Events: Eleven SAEs were reported during the course ofthe study. None of these was causally related to vaccination by theinvestigator.

Conclusion(s):

The Hib response after vaccination with DTPa-HBV-IPV/HibPLG wassignificantly higher as compared to DTPa-HBV-IPV/Hib and was notdifferent from that to the standalone Hib conjugate vaccine HIBERIX®.There was thus no observed Hib immune interference problem with the newformulation.

The response to the other antigens administered was similar to that toDTPa-HBV-IPV/Hib and to DTPa-HBV-IPV in terms of seroprotection ratesand vaccine responses. The other antigens seem to be compatible with PLGas a vaccine excipient. Although GMC titres against pertactin and IPVtype I fell for DTPa-HBV-IPV/HibPLG as compared with DTPa-HBV-IPV/Hib(they were, however, more similar to the titres for the DTPa-HBV-IPV+Hibgroup) the vaccine response to pertactin and the seroprotection ratesfor IPV type I for all vaccines were comparable.

The DTPa-HBV-IPV/HibPLG showed a reactogenicity comparable to that ofthe DTPa-HBV-IPV/Hib vaccine.

We claim:
 1. An immunogenic composition comprising a capsular polysaccharide or oligosaccharide of Haemophilus influenzae B (PRP), and a polyanionic polymer which is an oligo- or poly-peptide consisting of, on average, 8-117 residues and comprising anionic constitutional repeating units obtained from a group consisting of: L-aspartic acid, D-aspartic acid, L-glutamic acid, D-glutamic acid, and salts of the foregoing.
 2. The immunogenic composition of claim 1, wherein PRP is conjugated to a carrier protein which is a source of T-helper cell epitopes.
 3. The immunogenic composition of claim 2, wherein the carrier protein is selected from the group consisting of: tetanus toxoid, diphtheria toxoid, CRM197, and protein D.
 4. The immunogenic composition of claim 1, wherein the polyanionic polymer is an oligo- or poly-peptide which has a monomer content of 100% L-aspartic acid and/or L-glutamic acid.
 5. The immunogenic composition of claim 1, wherein the oligo- or polypeptide consists of, on average, 15-18 residues.
 6. The immunogenic composition of claim 1, wherein the polyanionic polymer is polyanionic heteropolymer.
 7. The immunogenic composition of claim 6, wherein the polyanionic heteropolymer consists of two distinct anionic constitutional repeating units.
 8. The immunogenic composition of claim 1, wherein the polyanionic polymer is a polyanionic homopolymer.
 9. The immunogenic composition of claim 1, wherein the polyanionic polymer is poly-L-glutamic acid (PLG).
 10. The immunogenic composition of claim 1, wherein the immunogenic composition comprises one or more further antigens.
 11. The immunogenic composition of claim 10, wherein the one or more further antigens comprise one or more meningococcal capsular oligosaccharide or polysaccharide-carrier protein conjugates selected from a group consisting of: MenC, MenY, MenA and MenW, preferably MenC and/or MenY.
 12. The immunogenic composition of claim 10, wherein the one or more further antigens comprise one or more pneumococcal capsular oligosaccharide or polysaccharide-carrier protein conjugates.
 13. The immunogenic composition of claim 11, wherein the carrier protein is selected from the group consisting of: tetanus toxoid, diphtheria toxoid, CRM197, and protein D.
 14. The immunogenic composition of claim 10, wherein the one or more further antigens comprise tetanus toxoid, diphtheria toxoid, and inactivated whole-cell B. pertussis or one or more acellular B. pertussis antigens.
 15. The immunogenic composition of claim 10, wherein the one or more further antigens comprise one or more acellular B. pertussis antigens selected from the group consisting of: pertussis toxiod, FHA, pertactin, agglutinogen 2 and agglutinogen
 3. 16. The immunogenic composition of claim 10, wherein the one or more further antigens comprise either or both of Inactivated Polio Vaccine (IPV) and Hepatitis B surface antigen, wherein Hepatitis B surface antigen is preferably adsorbed onto aluminium phosphate.
 17. The immunogenic composition of claim 1, which is lyophilised and further comprises a stabilizing excipient selected from the group consisting of: glucose, maltulose, iso-maltulose, lactulose, sucrose, sorbitol, maltose, lactose, iso-maltose, maltitol, lactitol, palatinit, trehalose, raffinose, stachyose, and melezitose; preferably sucrose.
 18. A vaccine comprising the immunogenic composition of claim 1 and a pharmaceutically acceptable excipient.
 19. A method of preventing or treating H. influenzae B disease comprising the step of administering a pharmaceutically effective amount of the vaccine of claim 18 to a patient in need thereof.
 20. The immunogenic composition of claim 12, wherein the carrier protein is selected from the group consisting of: tetanus toxoid, diphtheria toxoid, CRM197, and protein D. 